嗜酸氧化硫硫杆菌对黄铜矿浸出中钝化胁迫的生理应答机制

Passivation stress of jarosite/sulfur membrane can inhibit the iron/sulfur metabolism of bioleaching microorganism, and greatly restrict chalcopyrite bioleaching. After a direct long-term domestication, Acidithiobacillus thiooxidans CCTCC M2012104 showed a stronger ability in resisting passivation stress and a competitive advantage in population community. However, the physiological response mechanism under passivation stress is still not understood. In this research, this strain is employed as the model strain. With the assistance of pyrosequencing and oligonucleotide probe techniques, the effects of intervention of adapted A. thiooxidans in the original mixed-strains bioleaching system under passivation stress on the level of community structure of planktonic/attached cells is analyzed, then the synergistic mechanism is determined. The response mechanism of intracellular micro-environment is revealed via comparing the factor fluctuations of energy auxiliary, oxidation reduction balance status and electron transport complex. With the analysis of transcriptomics and proteomics, the key fundamental protein is explored and its regulatory mechanism for resisting passivation stress is revealed. Based on the above studies, the physiological response mechanism in resisting passivation stress will be illustrated from the multi-angels of intracellular physiology-fundamental protein-regulation mode-environmental adaptation in passivation stress. The project will help to understand the physiological mechanism of bioleaching microorganism in responding hostile environmental stress, which also provides a scientific basis for directly modifying bioleaching strain with the high ability in resisting passivation stress.

黄钾铁矾/硫膜钝化胁迫抑制浸矿菌的铁/硫代谢，严重制约黄铜矿生物浸出。经长期定向驯化的嗜酸氧化硫硫杆菌（Acidithiobacillus thiooxidans CCTCC M2012104），呈现较强抵御钝化胁迫的生理活性，种群竞争优势明显，但对钝化胁迫的生理应答机制尚不明晰。本项目以该菌为模式菌株，利用焦磷酸测序和寡核苷酸探针技术，分析钝化胁迫下驯化菌株干预原混菌浸出体系的过程，对浮游/附着细胞种群结构演替的影响及其协同机制；比较能量辅因子、氧化还原平衡和电子传递体等参数变化，揭示胞内微环境生理应答机制；通过转录组学和蛋白质组学分析，挖掘抵御钝化胁迫的关键功能蛋白，并解析其调控机制。基于以上研究，从胞内生理-功能蛋白-调控模式-钝化环境适应性的关联角度，阐明驯化菌株抵御钝化胁迫的生理应答机制。本项目有助于理解浸矿菌抵御恶劣环境的生理机制，并为定向改造高耐胁迫的浸矿菌种提供了科学依据。

贫黄铜矿浸出过程面临钝化胁迫，会制约其浸出效率。本研究以工业浸出液来源的高附着性能潜力Acidithiobacillus thiooxidans CCTCC M 2012104为研究对象，通过测序和分子探针技术分析贫黄铜矿浸出过程微生物群落演替规律，发现嗜酸性氧化硫硫杆菌对其附着细胞依赖性更强。定向调控微生物群落结构，在浸出中后期，可有效削弱黄钾铁矾和硫膜的钝化效应。因此考虑借助强酸削弱浸出过程钝化胁迫压力过程。嗜酸氧化硫硫杆菌胞内微环境生理参数表明，胞内pH稳定中性，具有较强耐酸能力。高效液相色谱分析发现胞内游离谷氨酸、天冬氨酸等含量增加。GC-MS分析胁迫水平下细胞膜不饱和脂肪酸含量提升，增强了细胞膜流动性。环丙烷脂肪酸（C19-cyc）含量上升，有利于维持致密膜结构。抗氧化酶（SOD、CAT）活性逐渐增强，以清除重金属和胁迫引起的胞内物质过氧化。采用RNA-Seq组学技术比较在浸出过程胁迫条件下全局性基因转录水平差异。功能注释显示转录差异主要涉及双组分系统（TCS）、硫代谢、中心碳代谢、细胞运动和趋化性等。硫代谢的硫氧化还原酶编码基因sor和硫氧化系统（Sox）相关基因soxABXYZ的转录水平显著上调，无机硫化物（RISCs）效率提升。激光共聚焦显微镜（CLSM）观察表明EPS形成和生物膜形成紧密相关。实时定量PCR（qRT-PCR）验证胁迫响应过程中关键基因（atpB, sor, flik, glnA, soxX, mdtA, flip, holA）差异表达，初步构建A. thiooxidans CCTCC M 2012104胁迫压力下响应机制。